肺动脉高压的啮齿动物模型的血流动力学特性
Summary
Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.
Abstract
Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease.
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.
Introduction
肺动脉高压（PAH）是炎性细胞浸润，平滑肌细胞增殖和内皮细胞凋亡相关联的肺血管的疾病。这些变化导致肺小动脉闭塞，随后导致右心室（RV）功能障碍和心脏衰竭。为了了解病理生理学的基本PAH PAH和右心衰竭，多个不同的模型，包括基因和药理学的模型，为研究这种疾病已经发展（综述别处1,2）。
这些模型的，最流行的是在小鼠低氧诱导（HX）PAH和野百合碱（MCT）和在大鼠SU5416缺氧（SuHx）模型。在小鼠HX模型，小鼠被暴露到4周缺氧（无论是常压或低压，对应于18000英尺的0.10的FiO2高度），与内侧扩散所得的发展，增加RV SYST奥利奇压力和RV肥大3的发展。 MCT在通过一个不明确的机制，然后导致PAH 4的发展在损伤肺的内皮细胞60毫克/公斤的结果的单剂量。 SU5416是血管内皮生长因子受体（VEGFR）1和2阻断剂的抑制剂，并用单次皮下注射60毫克/千克，随后通过暴露于慢性缺氧3周导致永久肺动脉高血压具有相似病理改变治疗到出现在人类疾病，闭塞性血管病变5的形成。在过去几年中，对肺动脉高血压几个转基因小鼠模型已被开发。这些包括敲除和骨形态发生蛋白受体2（BMPR2）的突变，如BMPR2基因突变在PAH的两个家族和特发性形式存在，血红素氧合酶1敲除和IL-6的过度表达（别处1,2-综述）。
PH这些不同的啮齿动物模型有不同程度的肺动脉高压，右心室肥厚和右心衰竭。而缺氧和各种转基因小鼠模型导致比任一大鼠模型1温和得多的PAH，它允许不同的基因突变和其相关的分子信号转导途径的测试。在MCT模型并导致严重的肺动脉高压，虽然MCT似乎是有毒的内皮细胞在多种组织4。该SuHx模型的特征是血管变化更加类似于在人类原发性肺动脉高压看出，虽然需要同时药理操纵和缺氧曝光。此外，在所有这些模型中，可能存在与PAH的发展相关的组织病理学变化，肺动脉压和右心室功能之间的断开。这是相对于人类疾病，其中通常有病理变化之间的比例关系，pulmon的严重性元高血压和右心衰竭的程度。因此，PH这些啮齿动物模型的综合表征是必需的，涉及到RV功能（通常通过超声心动图）的评估，血液动力学（心导管）和心脏组织病理学和肺（从组织收获）。
在这个协议中，我们描述了用于在大鼠和小鼠的PAH模型的血流动力学表征的基本技术。这些常规技术可以应用到右心室和肺血管的任何研究，并且不限于PAH的模型。通过超声心动图可视化RV是以大鼠相对简单，但在小鼠中更有挑战性，因为它们的大小和RV的复杂几何形状。此外，用于定量RV功能的一些替代物，比如TAPSE，肺动脉（PA）的加速时间和PA血流频谱开槽，不能很好的在人类验证，并用PU的相关评估只是弱lmonary高血压和血流动力学侵入RV功能。右心室血流动力学的确定是最好的一个封闭胸完成，以保持与灵感负胸内压力的影响，虽然开胸导尿使用阻抗导管允许压力容积（PV）的判定回路和更详细的血流动力学的表征。如同任何程序，开发经验与程序，是实验成功的关键。
Protocol
Representative Results
Discussion
The protocols outlined here describe a comprehensive characterization of hemodynamics and right ventricular function in rodent models of pulmonary hypertension. While right heart catheterization as described here is a terminal procedure, the mortality associated with echocardiography is minimal, which allows for screening and follow-up of disease progression. However, similar to patients with PH having markedly increased mortality with anesthesia17, in our experience, rats with severe PH do not tolerate anesth…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SR is supported by NIH K08HL114643, Gilead Research Scholars in Pulmonary Arterial Hypertension and a Burroughs Wellcome Fund Career Award for Medical Scientists.
Materials
| Vevo 2100 Imaging System (120V) | VisualSonics, inc. | VS-11945 | |
| Vevo 2100 Imaging Station | VisualSonics, inc. | ||
| High-frequency Mechanical Transducers | VisualSonics, inc. | MS250, MS550D, MS400 | |
| Ultrasound Gel Parker | Laboratories Inc. | 01-08 | |
| PowerLab 4/35 | ADInstruments | ML765 | |
| Labchart 8 | ADInstruments | ||
| BP transducer with stopcock and cable | ADInstruments | MLT1199 | |
| BP transducer calibration kit | ADInstruments | MLA1052 | |
| Mikro-Tip Pressure Catheter for mouse | Millar | SPR-1000 | Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat) |
| Mikro-Tip Pressure Catheter for rat | Millar | SPR-513 | Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat) |
| Millar Mikro-Tip ultra-miniature PV loop catheter for mice | Millar | PVR-1035 | Alternative catheter available from Scisense FT112 (mouse) |
| Millar Mikro-Tip ultra miniature PV loop catheter for rats | Millar | SPR-869 | Alternative catheter available from Scisense FT112 (mouse) |
| Millar PV system MPVS-300 | Millar | MPVS-300 | |
| 4-0 Silk Black Braid 100 Yard Spool | Roboz Surgical | SUT-15-2 | |
| 6-0 Silk Black Braid 100 Yard Spool | Roboz Surgical | SUT-14-1 | |
| Iris Scissors, Delicate, Integra Miltex | VWR | 21909-248 | |
| VWR Dissecting Scissors, Sharp/Blunt Tip | VWR | 82027-588 | |
| VWR Delicate Scissors, 4 1/2" | VWR | 82027-582 | |
| Two star Hemostats, Excelta | VWR | 63042-090 | |
| Neutral-buffered formalin | VWR | 89370-094 | |
| Crotaline | Sigma | C2401 | |
| SU5416 | Tocris Biosciences | 3037 | |
| 3.5X-45X Boom Stand Trinocular Zoom Stereo Microscope | AmScope | SM-3BX | |
| PE (Polyethylene Tubing)-10 | Braintree Scientific Inc | PE10 36 FT | |
| PE (Polyethylene Tubing)-50 | Braintree Scientific Inc | PE50 36 FT | |
| PE (Polyethylene Tubing)-60 | Braintree Scientific Inc | PE60 36 FT | |
| Tabletop Isoflurane Anesthesia Unit | Kent Scientific | ACV-1205S | |
| Surgisuite multi-functional surgical platform | Kent Scientific | Surgisuite | |
| Retractor set | Kent Scientific | SURGI-5002 | |
| Anesthesia induction chamber | VetEquip | 941443 | |
| Anesthesia Gas filter canister | Kent Scientific | ACV-2001 | |
| Rodent nose cone | VetEquip | 921431 |
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